особенности микробиоты кишечника у больных хронической

Оригинальные статьи
ìÑä 616.23/.24-002.2-06:616.34-008.87
ОСОБЕННОСТИ МИКРОБИОТЫ КИШЕЧНИКА У БОЛЬНЫХ ХРОНИЧЕСКОЙ
ОБСТРУКТИВНОЙ БОЛЕЗНЬЮ ЛЕГКИХ
Огородова Л.М.1, Говорун В.М.2, Федосенко С.В.1, Карнаушкина М.А.3, Салтыкова И.В.1,
Алексеев Д.Г.2, Кострюкова Е.С.2, Тяхт А.В.2, Попенко А.С.2
1
2
3
Ñèáèðñêèé ãîñóäàðñòâåííûé ìåäèöèíñêèé óíèâåðñèòåò, ã. Òîìñê
ÍÈÈ ôèçèêî-õèìè÷åñêîé ìåäèöèíû ÔÌÁÀ Ðîññèè, ã. Ìîñêâà
Ìîñêîâñêèé ãîñóäàðñòâåííûé ìåäèêî-ñòîìàòîëîãè÷åñêèé óíèâåðñèòåò èì. À.È. Åâäîêèìîâà, ã. Ìîñêâà
РЕЗЮМЕ
 ñòàòüå îáîáùåíû ðåçóëüòàòû èññëåäîâàíèé, ïîñâÿùåííûõ èçó÷åíèþ ñîñòàâà ñîîáùåñòâà ìèêðîîðãàíèçìîâ â îáðàçöàõ êàëà áîëüíûõ õðîíè÷åñêîé îáñòðóêòèâíîé áîëåçíüþ ëåãêèõ (ÕÎÁË) è çäîðîâûõ äîáðîâîëüöåâ ìåòîäîì ïîëíîãåíîìíîãî ìåòàãåíîìíîãî ñåêâåíèðîâàíèÿ. Ïîêàçàíî, ÷òî ñîîáùåñòâî ìèêðîîðãàíèçìîâ êèøå÷íèêà ó ïàöèåíòîâ ñ ÕÎÁË õàðàêòåðèçóåòñÿ ñòîëü æå ðàçíîîáðàçíûì òàêñîíîìè÷åñêèì ñîñòàâîì ìåòàãåíîìîâ, ÷òî è ìèêðîáèîòà çäîðîâûõ äîáðîâîëüöåâ. Ïðè
ýòîì íîðìàëüíûé ñîñòàâ êèøå÷íîé ìèêðîáèîòû ó áîëüíûõ ÕÎÁË ïîäâåðãàåòñÿ êà÷åñòâåííîé è
êîëè÷åñòâåííîé ìîäèôèêàöèè.  îòëè÷èå îò çäîðîâûõ äîáðîâîëüöåâ, êèøå÷íàÿ ìèêðîáèîòà ó áîëüíûõ ÕÎÁË õàðàêòåðèçóåòñÿ ïðèñóòñòâèåì òàêèõ ïðåäñòàâèòåëåé Proteobacteria, êàê Citrobacter,
Enterobacter, Eggerthella, Proteus, Salmonella, Anaerococcus, Clostridium difficile, Pseudomonas, à
òàêæå áîëåå âûñîêîé îáñåìåíåííîñòüþ ãðèáêàìè ðîäà Candida (Candida dubliniensis è Candida
albicans).
КЛЮЧЕВЫЕ СЛОВА: ñîîáùåñòâî ìèêðîîðãàíèçìîâ êèøå÷íèêà, êèøå÷íàÿ ìèêðîáèîòà, ïîëíîãå-
íîìíîå ìåòàãåíîìíîå ñåêâåíèðîâàíèå, ÕÎÁË.
Введение
Ìèêðîáíûå ïîïóëÿöèè èãðàþò âàæíóþ ðîëü â
ïîääåðæàíèè çäîðîâüÿ.  æåëóäî÷íî-êèøå÷íîì òðàêòå
îíè äåòåðìèíèðóþò ôîðìèðîâàíèå ëîêàëüíîãî è ñèñòåìíîãî èììóíèòåòà, ñïîñîáñòâóþò êèøå÷íîìó àíãèîãåíåçó è ÿâëÿþòñÿ âàæíûì ôàêòîðîì íîðìàëüíîãî
ïèùåâàðåíèÿ [1]. Ìèêðîáèîòà êèøå÷íèêà ÿâëÿåòñÿ
îäíîé èç íàèáîëåå èçó÷åííûõ áëàãîäàðÿ ïðèìåíåíèþ
ïðåèìóùåñòâåííî êóëüòóðàëüíûõ ìåòîäîâ, ÷òî, îäíàêî, íå äàåò âîçìîæíîñòè îöåíèòü âñå âèäîâîå ìíîãîîáðàçèå íàñåëÿþùåé êèøå÷íèê ìèêðîôëîðû â íîðìå è
íå ïîçâîëÿåò ïðîâåñòè ïîëíîöåííûé àíàëèç îñîáåííîñòåé èçìåíåíèÿ ñîîáùåñòâà ìèêðîîðãàíèçìîâ ïðè ïàòîëîãèè. Òàê, ñ ïîìîùüþ êóëüòóðàëüíûõ ìåòîäîâ âîçìîæíî âûÿâèòü íå áîëåå 1–10% áàêòåðèé. Êðîìå òîãî,
êóëüòèâèðîâàíèå ÿâëÿåòñÿ äîëãèì ïðîöåññîì äèàãíîñòèêè èíôåêöèé [2]. Âíåäðåíèå ñîâðåìåííûõ ìîëåêóëÿðíî-ãåíåòè÷åñêèõ ìåòîäîâ èäåíòèôèêàöèè ïîçâîëÿåò
ñóùåñòâåííî ðàñøèðèòü çíàíèÿ î êèøå÷íîì ìèêðî Ôåäîñåíêî Ñåðãåé Âÿ÷åñëàâîâè÷, e-mail: [email protected]
áèîìå è åãî ìîäèôèêàöèè ïðè ðàçëè÷íûõ ïàòîëîãè÷åñêèõ ñîñòîÿíèÿõ, ñîïðÿæåííûõ, íàïðèìåð, ñ ÷àñòûìè
êóðñàìè àíòèáèîòèêîòåðàïèè.
Õðîíè÷åñêàÿ îáñòðóêòèâíàÿ áîëåçíü ëåãêèõ
(ÕÎÁË) – ðàñïðîñòðàíåííîå ñðåäè âçðîñëîãî íàñåëåíèÿ çàáîëåâàíèå áðîíõî-ëåãî÷íîé ñèñòåìû, êîòîðîå
ïðèâîäèò ê ñóùåñòâåííîìó ñíèæåíèþ êà÷åñòâà æèçíè
è àññîöèèðîâàíî ñ ÷àñòûìè èíôåêöèîííî-çàâèñèìûìè
îáîñòðåíèÿìè. Â ñðåäíåì áîëüíîé ÕÎÁË òÿæåëîãî è
î÷åíü òÿæåëîãî òå÷åíèÿ ïåðåíîñèò â ãîä îò îäíîãî äî
÷åòûðåõ è áîëåå ýïèçîäîâ îáîñòðåíèÿ. Êàæäîå íîâîå
îáîñòðåíèå ÕÎÁË íå òîëüêî ïîâûøàåò ðèñê äîëãîñðî÷íîãî ñíèæåíèÿ ôóíêöèè âíåøíåãî äûõàíèÿ, íî è
îáóñëîâëèâàåò áîëåå âûñîêóþ ÷àñòîòó àíòèáèîòèêîòåðàïèè [3].  ñâîþ î÷åðåäü, íàðóøåíèå ðîñòà íîðìàëüíîé ìèêðîôëîðû êèøå÷íèêà íà ôîíå ïðèåìà àíòèáàêòåðèàëüíûõ ïðåïàðàòîâ ìîæåò ñïîñîáñòâîâàòü ïàòîëîãè÷åñêîé
êîëîíèçàöèè
åãî
ìèêðîîðãàíèçìàìè,
íàïðèìåð Clostridium èëè Candida Sðð. [4]. Äî íàñòîÿùåãî ìîìåíòà ãëóáèííûé àíàëèç ñîñòàâà ñîîáùåñòâ ìèêðîîðãàíèçìîâ, íàñåëÿþùèõ êèøå÷íóþ ìèêðîáèîòó, ó áîëüíûõ ÕÎÁË íå ïðîâîäèëñÿ.
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
55
Огородова Л.М., Говорун В.М., Федосенко С.В. и др.
Öåëü èññëåäîâàíèÿ – âûïîëíèòü ãëóáèííûé àíàëèç
òàêñîíîìè÷åñêîãî ñîñòàâà êèøå÷íîé ìèêðîáèîòû ó
áîëüíûõ ÕÎÁË è çäîðîâûõ äîáðîâîëüöåâ.
Материал и методы
Áûëè èññëåäîâàíû 52 îáðàçöà êàëà áîëüíûõ ÕÎÁË
ðàçëè÷íîé ñòåïåíè òÿæåñòè (ñðåäíèé âîçðàñò ïàöèåíòîâ – (58,22 ± 9,6) ãîäà). Ñðåäè íèõ 34 ïàöèåíòà ñ
ÕÎÁË ñðåäíåé ñòåïåíè òÿæåñòè è 18 ïàöèåíòîâ ñ
ÕÎÁË òÿæåëîãî è î÷åíü òÿæåëîãî òå÷åíèÿ. Ñòåïåíü
òÿæåñòè îïðåäåëÿëàñü â ñîîòâåòñòâèè ñî ñïèðîìåòðè÷åñêîé êëàññèôèêàöèåé GOLD (2010).  èññëåäîâàíèå
âêëþ÷àëèñü áîëüíûå ÕÎÁË ñòàáèëüíîãî òå÷åíèÿ ñ îòñóòñòâèåì àíàìíåçà îáîñòðåíèé è ïðèåìà àíòèáèîòèêîâ íà ïðîòÿæåíèè 3 ìåñ è áîëåå.
Ïîñëå âûäåëåíèÿ òîòàëüíîé ÄÍÊ áûëî âûïîëíåíî
ïîëíîãåíîìíîå (shotgun) ìåòàãåíîìíîå ñåêâåíèðîâàíèå
íà ïðèáîðàõ SOLiD 4 è SOLiD 5500W (Life Technologies, ÑØÀ). Ñðåäíåå ÷èñëî ðèäîâ íà îáðàçåö ñîñòàâèëî 38 ìëí øòóê. Ïðåäâàðèòåëüíàÿ ôèëüòðàöèÿ ðèäîâ
ïîêàçàëà, ÷òî êà÷åñòâî ñåêâåíèðîâàíèÿ ñîîòâåòñòâóåò
íîðìå ïî ïðîòîêîëó, äîëÿ îòôèëüòðîâàííûõ íèçêîêà÷åñòâåííûõ ðèäîâ ñîñòàâèëà (23,2 ± 0,7) %. Ñ öåëüþ
îïðåäåëåíèÿ òàêñîíîìè÷åñêîãî ñîñòàâà ìèêðîáèîòû
êèøå÷íèêà ðèäû áûëè îòîáðàæåíû íà ïðåäñòàâèòåëüíûé êàòàëîã, ñîäåðæàùèé 354 ãåíîìà êèøå÷íûõ áàêòåðèé. Äëÿ êàæäîãî áàêòåðèàëüíîãî òàêñîíà (âèäà,
ðîäà) åãî îòíîñèòåëüíàÿ äîëÿ â ÷èñëå âñåõ áàêòåðèé
áûëà âû÷èñëåíà èñõîäÿ èç ñóììàðíîé äëèíû ðèäîâ,
Особенности микробиоты кишечника у больных ХОБЛ
êàðòèðîâàâøèõñÿ íà ãåíîìû, ïðèíàäëåæàùèå ê òàêñîíó, è ïðèâåäåíà ê ïðîöåíòíîìó ïðåäñòàâëåíèþ. Äîëÿ
èäåíòèôèöèðîâàííûõ ðèäîâ ñîñòàâèëà (25,0 ± 12,5) %,
÷òî ñîîòâåòñòâóåò ïîïóëÿöèîííîé íîðìå ïî ïðîòîêîëó
ìåòàãåíîìíîãî àíàëèçà.
 êà÷åñòâå ãðóïïû ñðàâíåíèÿ (êîíòðîëü) èñïîëüçîâàëèñü ðåçóëüòàòû èññëåäîâàííûõ ïî àíàëîãè÷íîé ìåòîäèêå îáðàçöîâ êàëà, ïîëó÷åííûõ îò 96 çäîðîâûõ
äîáðîâîëüöåâ èç Ðîññèè [5].
Ñòàòèñòè÷åñêèé àíàëèç ïðîâîäèëñÿ íà ÿçûêå ïðîãðàììèðîâàíèÿ R 3.1.0. Äëÿ ïîäñ÷åòà ìàòðèöû ðàññòîÿíèé ìåæäó îáðàçöàìè áûëî èñïîëüçîâàíî ðàññòîÿíèå Áðýÿ-Êåðòèñà. Ïîèñê ïðèçíàêîâ, ïî êîòîðûì
ðàçëè÷àþòñÿ ãðóïïû îáðàçöîâ, âûïîëíÿëñÿ òåñòîì
Ìàííà–Óèòíè. Ïîïðàâêà íà ìíîæåñòâåííûå ñðàâíåíèÿ
ïðîèçâîäèëàñü ñ ïîìîùüþ FDR (False Discovery Rate).
Äëÿ ïîèñêà ðàçëè÷àþùèõñÿ íèçêîïðåäñòàâëåííûõ òàêñîíîâ áûëà èñïîëüçîâàíà áèáëèîòåêà metagenomeSeq
äëÿ ÿçûêà ïðîãðàììèðîâàíèÿ R.
Результаты и обсуждение
 õîäå àíàëèçà òàêñîíîìè÷åñêîãî ñîñòàâà ìåòàãåíîìíûõ îáðàçöîâ êàëà áîëüíûõ ÕÎÁË áûëè âûäåëåíû
ïðåîáëàäàþùèå òàêñîíû, ñîñòàâëÿþùèå 1% è áîëåå â
ñòðóêòóðå îáùåãî ÷èñëà áàêòåðèé õîòÿ áû â îäíîì
îáðàçöå. Âûäåëåííûå òàêñîíû ïðèâåäåíû â äèàãðàììàõ òàêñîíîìè÷åñêîãî ñîñòàâà îáðàçöîâ êàëà áîëüíûõ
ÕÎÁË íà óðîâíå ðîäîâ (ðèñ. 1) è âèäîâ (ðèñ. 2). Â äàííûõ ãèñòîãðàììàõ ñòðîêè ñîîòâåòñòâóþò îáðàçöàì
Ðèñ. 1. Òåïëîâàÿ êàðòà òàêñîíîìè÷åñêîãî ñîñòàâà îáðàçöîâ áîëüíûõ ÕÎÁË íà óðîâíå ðîäîâ
56
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
Оригинальные статьи
Ðèñ. 2. Òåïëîâàÿ êàðòà òàêñîíîìè÷åñêîãî ñîñòàâà îáðàçöîâ áîëüíûõ ÕÎÁË íà óðîâíå îòäåëüíûõ âèäîâ
(èåðàðõè÷åñêè êëàñòåðèçîâàíû ïî ñõîäñòâó ñîñòàâà), à
ñòîëáöû – áàêòåðèàëüíûì òàêñîíàì (ñëåäóþò â ïîðÿäêå óâåëè÷åíèÿ ñðåäíåé ïðåäñòàâëåííîñòè ïî âñåé âûáîðêå ñëåâà íàïðàâî).
 ïðåäñòàâëåííûõ ãèñòîãðàììàõ îáðàùàåò íà ñåáÿ
âíèìàíèå ÿâíàÿ êëàñòåðèçàöèÿ îáðàçöîâ ïî äâóì ãðóïïàì. Ïåðâàÿ ãðóïïà ñîñòîèò èç îáðàçöîâ ñ ïðåîáëàäàíèåì áàêòåðèé ðîäà Prevotella, à èìåííî Prevotella copri,
÷òî ñîîòâåòñòâóåò òàê íàçûâàåìîìó âòîðîìó ýíòåðîòèïó,
àññîöèèðîâàííîìó ñ äèåòîé, áîãàòîé ñëîæíûìè óãëåâîäàìè [6]. Âî âòîðîé ãðóïïå îáðàçöîâ ïðåîáëàäàþò áàêòåðèè ðîäà Bacteroides, ÷òî áîëåå õàðàêòåðíî äëÿ ëþäåé, ïðèäåðæèâàþùèõñÿ òàê íàçûâàåìîé çàïàäíîé äèåòû, áîãàòîé áåëêîì è æèâîòíûìè æèðàìè [6, 7].
Ãëóáèííûé àíàëèç òàêñîíîìè÷åñêîãî ñîñòàâà ìèêðîîðãàíèçìîâ â êàëå ïàöèåíòîâ ñ ÕÎÁË ïîçâîëèë âûäåëèòü ðÿä îáðàçöîâ, îòëè÷àþùèõñÿ âûñîêèì óðîâíåì
îáñåìåíåííîñòè ïðåäñòàâèòåëÿìè ãðóïïû Proteobacteria (òàáë. 1), áîëüøèíñòâî èç êîòîðûõ â íîðìå îòñóòñòâóþò èëè ñëàáî ïðåäñòàâëåíû â ìèêðîáèîòå êèøå÷íèêà. Òàê, íàïðèìåð, ñëåäóåò îòìåòèòü Citrobacter
(íàéäåíû â 11 îáðàçöàõ), Klebsiella (â 9 îáðàçöàõ),
Enterobacter (â 7 îáðàçöàõ), Eggerthella (â 8 îáðàçöàõ),
Proteus (â 2 îáðàçöàõ), Salmonella (â 6 îáðàçöàõ),
Anaerococcus (â 1 îáðàçöå), Clostridium difficile (â 7
îáðàçöàõ), Pseudomonas (â 1 îáðàçöå).
Òàáëèöà 1
ÉÛÔÔ‡ Proteobacteria, Ô‰ÒÚ‡‚ÎÂÌ̇fl ‚ Ó·‡Áˆ‡ı ͇· ·ÓθÌ˚ı ïéÅã
Êîëè÷åñòâî îáðàçöîâ
% â ñòðóêòóðå áèîìà
Citrobacter
Ðîä áàêòåðèé
11
0,03–0,93
Klebsiella
9
0,02–1,42
Enterobacter
7
0,01–1,57
Eggerthella
8
0,02–0,16
Proteus
2
0,05–2,30
Salmonella
6
0,02–0,13
Anaerococcus
1
0,05
Clostridiumdifficile
Pseudomonas
7
0,01–0,10
1
0,02
Ñ ïîìîùüþ òåñòà Ìàííà–Óèòíè áûëè âûÿâëåíû
ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ â ïðåäñòàâëåííîñòè
129 âèäîâ è 41 ðîäà ìåæäó 52 îáðàçöàìè ïàöèåíòîâ
è 96 îáðàçöàìè ìåòàãåíîìà êàëà çäîðîâûõ ëèö
(p < 0,05).  ÷àñòíîñòè, ñëåäóåò îòìåòèòü ïîíèæåííîå
ñîäåðæàíèå ïðåäñòàâèòåëåé îòäåëà Firmicutes (Faecalibacterium, Eubacterium) â ìåòàãåíîìå ïàöèåíòîâ ñ
ÕÎÁË. Áàêòåðèè ýòèõ òàêñîíîâ ïðîèçâîäÿò áóòèðàò,
òðàäèöèîííî ðàññìàòðèâàåìûé êàê ýíåðãåòè÷åñêèé
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
57
Íîðìàëèçîâàííîå çíà÷åíèå
ïðåäñòàâëåííîñòè (log)
0
2
4
6
8
ýêâèâàëåíò, ïîëó÷àåìûé îò êèøå÷íîé ìèêðîáèîòû è
áëàãîïðèÿòíûé äëÿ çäîðîâüÿ îðãàíèçìà.
Èçâåñòíî, ÷òî òåñò Ìàííà–Óèòíè íå ïîçâîëÿåò
äîñòîâåðíî ðàçëè÷àòü âûáîðêè ñ ìíîæåñòâîì íóëåâûõ
çíà÷åíèé, êàê áûâàåò äëÿ íèçêîïîêðûòûõ (ìàëî÷èñëåííûõ â îáðàçöå) îðãàíèçìîâ.  ñâÿçè ñ ýòèì â äîïîëíåíèå ê ñòàíäàðòíîìó ñòàòèñòè÷åñêîìó àíàëèçó
áûë ïðîâåäåí ãëóáèííûé àíàëèç ðàçëè÷èé òàêñîíîìè÷åñêîãî ñîñòàâà ìèêðîáèîòè÷åñêèõ ñîîáùåñòâ îáðàçöîâ êàëà ïàöèåíòîâ ñ ÕÎÁË è çäîðîâûõ ëèö ïóòåì
ïîñòðîåíèÿ ëèíåéíîé ðåãðåññèîííîé ìîäåëè ïî çíà÷åíèÿì êîëè÷åñòâà êàðòèðîâàâøèõñÿ ðèäîâ íà ðåôåðåíñíûå ãåíîìû ñ èñïîëüçîâàíèåì ïðîãðàììíîãî ïàêåòà
metagenomeSeq äëÿ ÿçûêà R [8].
 ðåçóëüòàòå ïðîâåäåííîãî àíàëèçà (metagenomeSeq) áûëè èäåíòèôèöèðîâàíû 110 âèäîâ ìèêðîîðãàíèçìîâ, ñòàòèñòè÷åñêè çíà÷èìî ðàçëè÷àþùèõñÿ ïî
ïðåäñòàâëåííîñòè â îáðàçöàõ áîëüíûõ ÕÎÁË è çäîðîâûõ äîáðîâîëüöåâ.  ÷àñòíîñòè, ýòèì ìåòîäîì áûëè
âûÿâëåíû ðàçëè÷èÿ â îáñåìåíåííîñòè ïðåäñòàâèòåëÿìè
Candida ìåæäó îáðàçöàìè îïûòíîé è êîíòðîëüíîé
âûáîðîê. Îñîáîãî âíèìàíèÿ çàñëóæèâàþò Candida
dubliniensis è Candida albicans, äëÿ êîòîðûõ îïðåäåëåíû
íàèáîëåå çíà÷èìûå ðàçëè÷èÿ ìåæäó ãðóïïàìè áîëüíûõ
ÕÎÁË è çäîðîâûõ äîáðîâîëüöåâ ïî çíà÷åíèþ êîýôôèöèåíòà íàêëîíà ïðÿìîé (âêëàäó ìèêðîîðãàíèçìà â ðàçëè÷èÿ ìåæäó ìåòàãåíîìàìè ñðàâíèâàåìûõ ãðóïï îáðàçöîâ) – 7,93 è 6,23 ñîîòâåòñòâåííî. È õîòÿ îïðåäåëÿåìîå
êîëè÷åñòâî ýòèõ ìèêðîîðãàíèçìîâ ìàëî (ïîêðûâàåòñÿ
ìåíåå 1% ãåíîìà), óêàçàííûé âûñîêî÷óâñòâèòåëüíûé
ìåòîä ïîçâîëÿåò âûÿâèòü ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ ìåæäó ãðóïïàìè: p ñ ïîïðàâêîé ïî FDR äëÿ
Candida dubliniensis ñîñòàâèëî 7,78E-11, äëÿ Candida
albicans – 2,12E-12. Íèæå ïðåäñòàâëåíû ðèñóíêè, äåìîíñòðèðóþùèå áîëåå âûñîêóþ îáñåìåíåííîñòü îáðàçöîâ êàëà áîëüíûõ ÕÎÁË øòàììîì Candida dubliniensis
CD36_1 (ðèñ. 3) è Candida albicans ïî ñðàâíåíèþ ñ îáðàçöàìè çäîðîâûõ äîáðîâîëüöåâ (ðèñ. 4).
Çäîðîâûå
Áîëüíûå ÕÎÁË
Ãðóïïû ñðàâíåíèÿ
Ðèñ. 3. Óðîâíè îáñåìåíåííîñòè Candida dubliniensis CD36_1
èññëåäóåìûõ îáðàçöîâ êàëà áîëüíûõ ÕÎÁË è êîíòðîëüíîé
âûáîðêè
58
Особенности микробиоты кишечника у больных ХОБЛ
Íîðìàëèçîâàííîå
çíà÷åíèå
Normalized log(cpt)
ïðåäñòàâëåííîñòè (log)
0.0
0.5
1.0
1.5
2.0
0
0,5
1,0
1,5
2,0
Огородова Л.М., Говорун В.М., Федосенко С.В. и др.
Çäîðîâûå
controls
Áîëüíûå
ÕÎÁË
patients
Ãðóïïû ñðàâíåíèÿ
Ðèñ. 4. Óðîâíè îáñåìåíåííîñòè Candida albicans W60 èññëåäóåìûõ îáðàçöîâ êàëà áîëüíûõ ÕÎÁË è êîíòðîëüíîé âûáîðêè
Çíà÷èìûå ðàçëè÷èÿ ïî ïðåäñòàâëåííîñòè â îáðàçöàõ êàëà áîëüíûõ ÕÎÁË è çäîðîâûõ äîáðîâîëüöåâ
áûëè âûÿâëåíû è ïî ðÿäó äðóãèõ ìèêðîîðãàíèçìîâ,
òðàäèöèîííî ðàññìàòðèâàåìûõ êàê ÷àñòü íîðìàëüíîé
ìèêðîáèîòû êèøå÷íèêà – Ruminococcus sp. è áèôèôèäîáàêòåðèè (â ÷àñòíîñòè, Bifidobacterium angulatum,
Bifidobacterium catenulatum DSM 16992, Bifidobacterium adolescentis ATCC 15703). Äàííûå ìèêðîîðãàíèçìû õàðàêòåðèçîâàëèñü îòðèöàòåëüíûì êîýôôèöèåíòîì
íàêëîíà ïðÿìîé, ò.å. èõ êîëè÷åñòâî áûëî ìåíüøå â
îáðàçöàõ ïàöèåíòîâ ñ ÕÎÁË â ñðàâíåíèè ñ îáðàçöàìè
çäîðîâûõ äîáðîâîëüöåâ (òàáë. 2, ðèñ. 5–7).
Òàáëèöà 2
åËÍÓÓ„‡ÌËÁÏ˚, ÒÚ‡ÚËÒÚ˘ÂÒÍË Á̇˜ËÏÓ ‡Á΢‡˛˘ËÂÒfl ÔÓ
Ô‰ÒÚ‡‚ÎÂÌÌÓÒÚË ‚ Ó·‡Áˆ‡ı ͇· ÏÂÊ‰Û „ÛÔÔ‡ÏË ·ÓθÌ˚ı ïéÅã
Ë Á‰ÓÓ‚˚ı ‰Ó·Ó‚ÓθˆÂ‚
Øòàìì ìèêðîîðãàíèçìîâ
Bifidobacterium pseudocatenulatum
DSM_20438
Êîýôôèöèåíò
íàêëîíà ïðÿìîé
adjPvalue
–3,473631472
0,00013926
Collinsella aerofaciens ATCC_25986
Bifidobacterium longum subsp. longum
JDM301
–3,806589893
0,000199063
–4,400957447
0,000104655
Bifidobacterium bifidum PRL2010
–4,779010156
7,32871E-05
Bifidobacterium breve DSM_20213
–4,97857245
6,62285E-06
Bifidobacterium angulatum DSM_20098
Bifidobacterium catenulatum
DSM_16992
Bifidobacterium adolescentis
ATCC_15703
Ruminococcus sp. 5_1_39BFAA
–5,871980832
6,22372E-05
–6,52313012
1,72973E-13
–7,237183311
0,00013926
–7,378799539
0,000199063
Èçâåñòíî, ÷òî ïðè ðàçëè÷íûõ âîñïàëèòåëüíûõ
çàáîëåâàíèÿõ æåëóäî÷íî-êèøå÷íîãî òðàêòà, à òàêæå
ó ëèö, ïðèìåíÿþùèõ àíòèáèîòèêè èëè ïîëó÷àþùèõ
õèìèîòåðàïèþ, íàáëþäàåòñÿ ñíèæåíèå òàêñîíîìè÷åñêîãî ðàçíîîáðàçèÿ êèøå÷íîé ìèêðîáèîòû (àëüôàðàçíîîáðàçèå). Ìåíåå ðàçíîîáðàçíàÿ ìèêðîáèîòà èìååò
ìåíüøèé ìåòàáîëè÷åñêèé ïîòåíöèàë è õàðàêòåðèçóåòñÿ
ñíèæåííîé óñòîé÷èâîñòüþ ê âîçäåéñòâèþ ïàòîãåíîâ.
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
Оригинальные статьи
Íîðìàëèçîâàííîå çíà÷åíèå
ïðåäñòàâëåííîñòè (log)
0 4 6 8
12
Ó÷èòûâàÿ, ÷òî ÕÎÁË ÿâëÿåòñÿ õðîíè÷åñêèì çàáîëåâàíèåì ñ âûñîêèì ïîòåíöèàëüíûì ðèñêîì ðàçâèòèÿ
îáîñòðåíèé è, ñëåäîâàòåëüíî, ïîòåíöèàëüíî áîëåå
âûñîêîé ÷àñòîòîé àíòèáèîòèêîòåðàïèè äëÿ èõ êóïèðîâàíèÿ, ìû ïðåäïîëîæèëè âîçìîæíîñòü ñíèæåíèÿ àëüôà-ðàçíîîáðàçèÿ â îáðàçöàõ êàëà áîëüíûõ ÕÎÁË â
ñðàâíåíèè ñ ãðóïïîé êîíòðîëÿ. Îäíàêî ñòàòèñòè÷åñêè
çíà÷èìûå ðàçëè÷èÿ ïî èíäåêñó àëüôà-ðàçíîîáðàçèÿ
îòñóòñòâîâàëè (p-value (U-test) = 0,52) – êèøå÷íàÿ
ìèêðîáèîòà áîëüíûõ ÕÎÁË õàðàêòåðèçîâàëàñü ñòîëü
æå ðàçíîîáðàçíûì òàêñîíîìè÷åñêèì ñîñòàâîì ìåòàãåíîìîâ, ÷òî è ìèêðîáèîòà çäîðîâûõ äîáðîâîëüöåâ.
Çäîðîâûå
Áîëüíûå ÕÎÁË
Ãðóïïû ñðàâíåíèÿ
Íîðìàëèçîâàííîå çíà÷åíèå
ïðåäñòàâëåííîñòè (log)
4 6 8 8 12
16
Ðèñ. 5. Óðîâíè îáñåìåíåííîñòè Ruminococcus sp. 5_1_39BFAA
èññëåäóåìûõ îáðàçöîâ êàëà áîëüíûõ ÕÎÁË è êîíòðîëüíîé
âûáîðêè
Çäîðîâûå
Áîëüíûå ÕÎÁË
Ãðóïïû ñðàâíåíèÿ
Íîðìàëèçîâàííîå çíà÷åíèå
ïðåäñòàâëåííîñòè (log)
0 2 4 6 8
12
Ðèñ. 6. Óðîâíè îáñåìåíåííîñòè Prevotella copri DSM_18205 èññëåäóåìûõ îáðàçöîâ êàëà áîëüíûõ ÕÎÁË è êîíòðîëüíîé
âûáîðêè
Çäîðîâûå
Áîëüíûå ÕÎÁË
Ãðóïïû ñðàâíåíèÿ
Ðèñ. 7. Óðîâíè îáñåìåíåííîñòè Bifidobacterium adolescentis
ATCC_15703 èññëåäóåìûõ îáðàçöîâ êàëà áîëüíûõ ÕÎÁË è êîíòðîëüíîé âûáîðêè
Ñëåäóåò îòìåòèòü îäèí îáðàçåö èç ãðóïïû áîëüíûõ
ÕÎÁË, äëÿ êîòîðîãî çíà÷åíèå èíäåêñà àëüôà-ðàçíîîáðàçèÿ ñîñòàâèëî 0,11. Äàííûé îáðàçåö õàðàêòåðèçîâàëñÿ ðåêîðäíûì ñîäåðæàíèåì ìèêðîîðãàíèçìîâ ðîäà
Bacteroides – 98,5%.
Заключение
Îáîáùàÿ ïîëó÷åííûå ðåçóëüòàòû, ìîæíî ñäåëàòü
íåñêîëüêî âûâîäîâ.
1. Ñîîáùåñòâî ìèêðîîðãàíèçìîâ êèøå÷íèêà ó ïàöèåíòîâ ñ ÕÎÁË õàðàêòåðèçóåòñÿ ñòîëü æå ðàçíîîáðàçíûì òàêñîíîìè÷åñêèì ñîñòàâîì ìåòàãåíîìîâ, ÷òî è
ìèêðîáèîòà çäîðîâûõ äîáðîâîëüöåâ.
2. Ñîñòàâ êèøå÷íîé ìèêðîáèîòû ó áîëüíûõ ÕÎÁË
ïîäâåðãàåòñÿ êà÷åñòâåííîé è êîëè÷åñòâåííîé ìîäèôèêàöèè. Òàê, âûÿâëåíû ñòàòèñòè÷åñêè çíà÷èìûå ðàçëè÷èÿ â ïðåäñòàâëåííîñòè áîëåå 42 ðîäîâ è 110 âèäîâ
ìèêðîîðãàíèçìîâ â îáðàçöàõ êàëà áîëüíûõ ÕÎÁË è
çäîðîâûõ äîáðîâîëüöåâ. Îäíàêî äàííûå ðàçëè÷èÿ íå
ÿâëÿþòñÿ ñòîëü çíà÷èòåëüíûìè, ÷òîáû âûäåëèòü èçó÷åííûå îáðàçöû â îòäåëüíûé âàðèàíò êèøå÷íîé ìèêðîáèîòû.
3. Êèøå÷íàÿ ìèêðîáèîòà áîëüíûõ ÕÎÁË õàðàêòåðèçóåòñÿ íàëè÷èåì îòñóòñòâóþùèõ èëè ñëàáî ïðåäñòàâëåííûõ â íîðìå ïðåäñòàâèòåëåé Proteobacteria,
òàêèõ êàê Citrobacter, Enterobacter, Eggerthella, Proteus, Salmonella, Anaerococcus, Clostridium difficile,
Pseudomonas, à òàêæå áîëåå âûñîêîé îáñåìåíåííîñòüþ
ãðèáêàìè ðîäà Candida (Candida dubliniensis è Candida albicans).
4. Â ñðàâíåíèè ñ ñîîáùåñòâîì ìèêðîîðãàíèçìîâ
çäîðîâûõ ëèö êèøå÷íàÿ ìèêðîáèîòà áîëüíûõ ÕÎÁË
õàðàêòåðèçóåòñÿ ñíèæåíèåì ïðåäñòàâëåííîñòè ìèêðîîðãàíèçìîâ Firmicutes (Faecalibacterium, Eubacterium),
Ruminococcus sp., áèôèäîáàêòåðèé (Bifidobacterium angulatum, Bifidobacterium catenulatum, Bifidobacterium adolescentis), êîòîðûå òðàäèöèîííî ðàññìàòðèâàþòñÿ êàê
÷àñòü íîðìàëüíîé ìèêðîôëîðû êèøå÷íèêà.
Õðîíè÷åñêàÿ îáñòðóêòèâíàÿ áîëåçíü ëåãêèõ – çàáîëåâàíèå, õàðàêòåðèçóþùååñÿ âûñîêèì ðèñêîì îáîñòðåíèé ñ ïîòðåáíîñòüþ â àíòèáèîòèêîòåðàïèè. Âåðîÿòíî, ÷àñòûå, íåðàöèîíàëüíûå êóðñû ïðèåìà àíòèáàêòåðèàëüíûõ ïðåïàðàòîâ, âëèÿþùèõ íà íîðìàëüíóþ
êèøå÷íóþ ìèêðîôëîðó, ìîãóò êà÷åñòâåííî è êîëè÷åñòâåííî ìîäèôèöèðîâàòü ñîñòàâ ñîîáùåñòâà ìèêðîîðãàíèçìîâ êèøå÷íèêà.
Ðàáîòà âûïîëíåíà ïðè ïîääåðæêå Ìèíèñòåðñòâà
îáðàçîâàíèÿ è íàóêè Ðîññèéñêîé Ôåäåðàöèè â ðàìêàõ
Ôåäåðàëüíîé öåëåâîé ïðîãðàììû «Èññëåäîâàíèÿ è ðàçðàáîòêè ïî ïðèîðèòåòíûì íàïðàâëåíèÿì ðàçâèòèÿ íàó÷íîòåõíîëîãè÷åñêîãî êîìïëåêñà Ðîññèè íà 2014–2020 ãã.»
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
59
Огородова Л.М., Говорун В.М., Федосенко С.В. и др.
(ÃÊ ¹ 14.604.21.0075, óíèêàëüíûé èäåíòèôèêàòîð
RFMEFI60414X0075).
Литература
1. Abt M.C., Artis D. The intestinal microbiota in health
and disease: the influence of microbial products on immune cell homeostasis // Curr. Opin. Gastroenterol. 2009.
25. P. 496–502.
2. Staley J.T., Konopka A. Measurement of in situ activities of
nonphotosynthetic microorganisms in aquatic and terrestrial
habitats // Annu. Rev. Microbiol. 1985. 39. P. 321–346.
3. Global Initiative for Chronic Obstructive Lung Disease
(GOLD). URL: http://www.goldcopd.org/uploads/users/files/GOLD_Report_2011_Feb21.pdf
4. Hooper L.V., Gordon J.I. Commensal host-bacterial relationships in the gut // Science. 2001. 292. P. 1115–1118.
Особенности микробиоты кишечника у больных ХОБЛ
5. Tyakht A.V., Kostryukova E.S., Popenko A.S., Belenikin M.S.,
Pavlenko A.V., Larin A.K., Govorun V.M. Human gut microbiota community structures in urban and rural populations in Russia // Nat. Commun. 2013. 4.
6. De Filippo C., Cavalieri D., Di Paola M., Ramazzotti M.,
Poullet J.B., Massart S., Collini S., Pieraccini G., Lionetti P.
Impact of diet in shaping gut microbiota revealed by a
comparative study in children from Europe and rural Africa
// Proc. Natl. Acad. Sci. USA. 2010. 107 (33). P. 14691–
14696.
7. Arumugam Ì., Raes J., Pelletier E., Le Paslier D. et al.
Enterotypes of the human gut microbiome // Nature. 2011.
12, 473 (7346). P. 174–180.
8. Joseph N., Paulson O., Colin Stine, Héctor Corrada Bravo,
Mihai Pop. Differential abundance analysis for microbial
marker-gene surveys // Nat. Methods. 2013. 10 (12).
P. 1200–1202.
Ïîñòóïèëà â ðåäàêöèþ 10.09.2014 ã.
Óòâåðæäåíà ê ïå÷àòè 09.10.2014 ã.
é„ÓÓ‰Ó‚‡ ã˛‰ÏË· åËı‡ÈÎӂ̇ – ä-ð ìåä. íàóê, ïðîôåññîð, ÷ëåí-êîððåñïîíäåíò ÐÀÌÍ, çàâ. êàôåäðîé ôàêóëüòåòñêîé ïåäèàòðèè ñ êóðñîì
äåòñêèõ áîëåçíåé ëå÷åáíîãî ôàêóëüòåòà ÑèáÃÌÓ (ã. Òîìñê), çàìåñòèòåëü ìèíèñòðà íàóêè è îáðàçîâàíèÿ Ðîññèéñêîé Ôåäåðàöèè.
ÉÓ‚ÓÛÌ Ç‡‰ËÏ å‡Íӂ˘ – ä-ð áèîë. íàóê, ïðîôåññîð, ÷ëåí-êîððåñïîíäåíò ÐÀÍ, çàìåñòèòåëü äèðåêòîðà ïî íàó÷íîé ðàáîòå ÍÈÈ ôèçèêîõèìè÷åñêîé ìåäèöèíû ÔÌÁÀ Ðîññèè (ã. Ìîñêâà).
î‰ÓÒÂÌÍÓ ëÂ„ÂÈ Çfl˜ÂÒ·‚ӂ˘ () – êàíä. ìåä. íàóê, äîêòîðàíò êàôåäðû ãîñïèòàëüíîé òåðàïèè ñ êóðñîì ôèçè÷åñêîé ðåàáèëèòàöèè è ñïîðòèâíîé ìåäèöèíû ÑèáÃÌÓ (ã. Òîìñê).
ä‡̇ۯÍË̇ å‡Ëfl ÄÎÂÍ҇̉ӂ̇ – êàíä. ìåä. íàóê, àññèñòåíò êàôåäðû ïóëüìîíîëîãèè ÔÏÄÎ ÌÃÌÑÓ èì. À.È. Åâäîêèìîâà (ã. Ìîñêâà).
ë‡ÎÚ˚ÍÓ‚‡ àË̇ Ç·‰ËÏËӂ̇ – êàíä. ìåä. íàóê, ìë. íàó÷. ñîòðóäíèê Öåíòðàëüíîé íàó÷íî-èññëåäîâàòåëüñêîé ëàáîðàòîðèè ÑèáÃÌÓ
(ã. Òîìñê).
ÄÎÂÍÒ‚ ÑÏËÚËÈ Éηӂ˘ – êàíä. áèîë. íàóê, çàâ. ëàáîðàòîðèåé áèîèíôîðìàòèêè ÍÈÈ ôèçèêî-õèìè÷åñêîé ìåäèöèíû ÔÌÁÀ Ðîññèè
(ã. Ìîñêâà).
äÓÒÚ˛ÍÓ‚‡ ÖÎÂ̇ ëÂ„‚̇ – êàíä. áèîë. íàóê, çàâ. ëàáîðàòîðèåé ïîñòãåíîìíûõ èññëåäîâàíèé â áèîëîãèè ÍÈÈ ôèçèêî-õèìè÷åñêîé ìåäèöèíû
ÔÌÁÀ Ðîññèè (ã. Ìîñêâà).
íflıÚ ÄÎÂÍ҇̉ ÇËÍÚÓӂ˘ – ìë. íàó÷. ñîòðóäíèê ëàáîðàòîðèè áèîèíôîðìàòèêè ÍÈÈ ôèçèêî-õèìè÷åñêîé ìåäèöèíû ÔÌÁÀ Ðîññèè
(ã. Ìîñêâà).
èÓÔÂÌÍÓ ÄÌ̇ ëÂ„‚̇ – àñïèðàíò, ëàáîðàíò ëàáîðàòîðèè áèîèíôîðìàòèêè ÍÈÈ ôèçèêî-õèìè÷åñêîé ìåäèöèíû ÔÌÁÀ Ðîññèè (ã. Ìîñêâà).
î‰ÓÒÂÌÍÓ ëÂ„ÂÈ Çfl˜ÂÒ·‚ӂ˘, e-mail: [email protected]
60
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
Оригинальные статьи
FEATURES OF THE INTESTINAL MICROBIOTA IN PATIENTS WITH CHRONIC
OBSTRUCTIVE PULMONARY DISEASE
Ogorodova L.M.1, Govorun V.M.2, Fedosenko S.V.1, Karnaushkina M.A.3, Saltykova I.V.1,
Alekseyev D.G.2, Kostryukova Ye.S.2, Tyakht A.V.2, Popenko A.S.2
1
2
3
Siberian State Medical University, Tomsk, Russian Federation
Research Institute for Physico-Chemical Medicine, FMBA, Moscow, Russian Federation
A.I. Yevdokimov Moscow State University of Medicine and Dentistry, Moscow, Russian Federation
ABSTRACT
The article summarizes the results of studies on the composition of microbial communities in stool samples of patients with chronic obstructive pulmonary disease (COPD) compared with healthy volunteers
using genome-metagenomic sequencing. It is shown that the microbial community of the intestine in
COPD patients is characterized as diverse taxonomic composition of metagenomes that the healthy volunteers microbiota. In this case, the normal composition of intestinal microbiota in patients with COPD
is exposed to the qualitative and quantitative modifications. In contrast to healthy volunteers, the intestinal microbiota in patients with COPD is characterized by the presence of representatives of the Proteobacteria, as Citrobacter, Enterobacter, Eggerthella, Proteus, Salmonella, Anaerococcus, Clostridium
difficile, Pseudomonas, as well as higher insemination fungus genus Candida (Candida dubliniensis and
Candida albicans).
KEY WORDS: community of microorganisms of the intestine, the intestinal microbiota, genome-
metagenomic sequencing, COPD.
Bulletin of Siberian Medicine, 2014, vol. 13, no. 5, pp. 55–61
References
1. Abt M.C., Artis D. The intestinal microbiota in health and disease: the influence of microbial products on immune cell homeostasis. Curr Opin Gastroenterol, 2009, 25, pp. 496–502.
2. Staley J.T., Konopka A. Measurement of in situ activities of
nonphotosynthetic microorganisms in aquatic and terrestrial
habitats. Annu. Rev. Microbiol., 1985, 39, pp. 321–346.
3. Global Initiative for Chronic Obstructive Lung Disease
(GOLD). URL: http://www.goldcopd.org/uploads/users/files/GOLD_Report_2011_Feb21.pdf
4. Hooper L.V., Gordon J.I. Commensal host-bacterial relationships in the gut. Science, 2001, 292, pp. 1115–1118.
5. Tyakht A.V., Kostryukova E.S., Popenko A.S., Belenikin
M.S., Pavlenko A.V., Larin A.K., Govorun V.M. Human
gut microbiota community structures in urban and rural
populations in Russia. Nat Commun, 2013, 4.
6. De Filippo C., Cavalieri D., Di Paola M., Ramazzotti M.,
Poullet J.B., Massart S., Collini S., Pieraccini G., Lionetti
P. Impact of diet in shaping gut microbiota revealed by a
comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. USA, 2010, 107 (33),
pp. 14691–14696.
7. Arumugam Ì., Raes J., Pelletier E., Le Paslier D. et al. Enterotypes of the human gut microbiome. Nature, 2011, 12,
473 (7346), pp. 174–180.
8. Joseph N., Paulson O., Colin Stine, Héctor Corrada Bravo,
Mihai Pop. Differential abundance analysis for microbial
marker-gene surveys. Nat. Methods, 2013, 10 (12),
pp. 1200–1202.
Ogorodova Lyudmila M., Siberian State Medical University, Tomsk, Russian Federation.
Govorun Vadim M., Research Institute for Physico-Chemical Medicine, FMBA, Moscow, Russian Federation.
Fedosenko Sergei V., Siberian State Medical University, Tomsk, Russian Federation.
Karnaushkina Mariya A., A.I. Yevdokimov Moscow State University of Medicine and Dentistry, Moscow, Russian Federation.
Saltykova Irina V., Siberian State Medical University, Tomsk, Russian Federation.
Alekseyev Dmitry G., Research Institute for Physico-Chemical Medicine, FMBA, Moscow, Russian Federation.
Kostryukova Yelena S., Research Institute for Physico-Chemical Medicine, FMBA, Moscow, Russian Federation.
Tyakht Aleksanr V., Research Institute for Physico-Chemical Medicine, FMBA, Moscow, Russian Federation.
Popenko Anna S., Research Institute for Physico-Chemical Medicine, FMBA, Moscow, Russian Federation.
Fedosenko Sergei V., e-mail: [email protected]
Бюллетень сибирской медицины, 2014, том 13, № 5, с. 55–61
61